Much of our understanding of cholinergic neurotransmission and synaptic function comes from studies in the peripheral nervous system. In the mammalian central nervous system (CNS), presumptive cholinergic pathways have been mapped extensively using histochemical techniques, and there exists sound evidence for identification of acetylcholine (ACh) as a CNS neurotransmitter. Toward improved understanding of central cholinergic mechanisms, the potentially promising application of curaremimetic neurotoxins as specific ligands for CNS nicotinic acetylcholine receptors (nAChR) is compromised by uncertainty regarding the physiological relevance of CNS toxin binding sites. The objective of the proposed research are threefold and sequential: 1) to develop further the characterization of specific toxin binding sites in the CNS, 2) to identify specific ligands for authentic CNS nAChR, and 3) to generate assays for CNS nAChR function. Relationships between toxin binding sites and sites that interact with other cholinergic ligands, affinity reagents, and antibodies directed against nAChR from the periphery will be ascertained first on the basis of radioligand binding properties and competition potency profiles. Membrane fragments rich in toxin/ligand/antibody sites will be prepared by density gradient centrifugation and affinity isolation techniques, and characterized physically and chemically. Integral model systems will be developed to test pharmacological actions of toxins and other ligands through their respective macromolecular sites in natural or reconstituted membrane vesicles and in tissue slices. Criteria for cholinergic receptor function will include chemical detection of agonist-induced transmembrane ion fluxes. Demonstration of pharmacological potency of toxins, ligands or antibodies, consistent with biochemical measures of receptor occupancy, will be suggestive of their physiological relevance as markers for authentic CNS nAChR.